Steroidal affinity labels of the estrogen receptor alpha. 4. Electrophilic 11beta-aryl derivatives of estradiol.

Ten electrophilic estradiol 11beta-aryl derivatives were synthesized, with three different types of 11beta-substituent: (i) pOO(CH(2))(2)X (compounds: 6, X = OSO(2)CH(3); 7, X = I; 13, X = NHCOCH(2)Cl; 15, X = N(CH(3))COCH(2)Br; and 16, X = N(CH(3))COCH(2)Cl); (ii) pOO(CH(2))(5)X (compounds: 17, X = I; 20, X = NHCOCH(2)Br; and 22, X = N(CH(3))COCH(2)Br); and (iii) pOC(triple bond)CCH(2)X (compounds: 27, X = NHCOCH(2)Cl; and 29, X = N(CH(3))COCH(2)Cl). The range of their apparent affinity constants for binding the lamb uterine estrogen receptor alpha (ERalpha) was 3-40% that of estradiol. Six electrophiles, chloroacetamides 13, 16, 27, and 29, iodide 17, and bromoacetamide 20 (whose arm linking the electrophilic carbon to the 11beta-phenyl group includes at least six bonds), were able to irreversibly inhibit the binding of [(3)H]estradiol to ER (25-60% decrease in binding sites), with the following compound effectiveness order: 17 < 13 < 16 approximately 20 approximately 27 approximately 29. Mesylate 6, iodide 7 (whose linking arm includes only three bonds), and bromoacetamides 15 and 22 (which differ from 16 by the Cl to Br change and from 20 by the NH to NCH(3) change, respectively) were much less effective (<10% decrease in binding sites, if any). The fact that the inactivation of estradiol-binding sites by the six electrophiles was totally prevented by estradiol indicated that they were ER affinity labeling agents. When ER was modified by methyl methanethiosulfonate, an SH-specific reagent, the different compounds led to very contrasting results in ER affinity labeling. With modified ER, iodide 17 and chloroacetamides 27 and 29 were practically inactive, chloroacetamides 13 and 16 and bromoacetamide 20 were still active but less effective than on the native ER, whereas tertiary bromoacetamides 15 and 22, found to be practically inactive on native ER, became the most effective electrophiles ( approximately 45% and approximately 65% binding sites inactivated, respectively). The results indicate that in the steroid-filled hormone-binding pocket: (i) nucleophilic residues are localized on the beta-side but relatively remote from the steroid nucleus (distance from C-11 > "seven bonds"); (ii) relatively discrete changes in the electrophilic functionality, such as Cl to Br or NH to NCH(3) of haloacetamido compounds, can markedly modify the positioning of the electrophilic center which could no longer react with the nucleophilic residues; and (iii) cysteine residues (probably homologues of human ERalpha cysteine 381 and/or cysteine 530) are, at least partly, the covalent attachment sites of the electrophiles. Moreover, modification of cysteine residues by methyl methanethiosulfonate changes the structure of the hormone-binding pocket, whose labeling by the various electrophiles is profoundly altered.

Steroidal affinity labels of the estrogen receptor. 3. Estradiol 11 beta-n-alkyl derivatives bearing a terminal electrophilic group: antiestrogenic and cytotoxic properties.

With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six electrophilic 11 beta-ethyl (C2), 11 beta-butyl (C4), or 11 beta-decyl (C10) derivatives of estradiol bearing an 11 beta-terminal electrophilic functionality, i.e. bromine (C4), (methylsulfonyl)oxy (C2 and C4), bromoacetamido (C2 and C4), and (p-tolylsulfonyl)oxy (C10), were synthesized. The range of their affinity constants for binding the estrogen receptor was 0.4-37% that of estradiol; the order of increasing affinity (i) relative to the 11 beta-alkyl arm was ethyl < butyl and (ii) relative to the electrophilic functionality was bromoacetamido < bromine < (methylsulfonyl)oxy. Regardless of the conditions used, including prolonged exposure of the receptor to various pH levels (7-9) and temperatures (0-25 degrees C), the extent of receptor affinity labeling by the 11 beta-ethyl and 11 beta-butyl compounds, if any, was under 10%. This was in sharp contrast to results obtained using 11 beta-((tosyloxy)decyl)estradiol which labeled from 60% to 90% of the receptor hormone-binding sites with an EC50 of approximately 10 nM. Estrogenic and antiestrogenic activities of the compounds were determined using the MVLN cell line, which was established from the estrogen-responsive mammary tumor MCF-7 cells by stable transfection of a recombinant estrogen-responsive luciferase gene. The two 11 beta-ethyl compounds were mainly estrogenic, whereas the three 11 beta-butyl and the 11 beta-decyl compounds essentially showed antiestrogenic activity. The fact that the chemical reactivities of 11 beta-ethyl and 11 beta-butyl compounds were not compromised by interaction with the estrogen receptor made the synthesized high-affinity compounds potential cytotoxic agents which might be able to exert either (i) a specific action on estrogen-regulated genes or (ii) a more general action in estrogen-target cells. Therefore the ability of the compounds (1) to irreversibly abolish estrogen-dependent expression of the luciferase gene and (2) to affect the proliferation of MVLN cells were determined. All electrophiles were able to irreversibly suppress expression of the luciferase gene; the antiestrogenic electrophiles were more potent than the estrogenic ones but less efficient than 4-hydroxytamoxifen, a classical and chemically inert triphenylethylene antiestrogen. Only the antiestrogenic electrophiles decreased cell proliferation; however, they were less potent than 4-hydroxytamoxifen. In conclusion, the synthesized electrophilic estradiol 11 beta-ethyl and 11 beta-butyl derivatives (i) were not efficient affinity labels of the estrogen receptor and (ii) did not display significant cytotoxicity in estrogen-sensitive mammary tumor cells. However, since these derivatives displayed high affinity for the estrogen receptor, they could be used to prepare potential cytotoxic agents which might be selective for tumors affecting estrogen-target tissues, by coupling them with a toxic moiety.

RU 58668: further in vitro and in vivo pharmacological data related to its antitumoral activity.

Previous studies with the pure antiestrogen RU 58668 showed that this compound proved to be highly antiproliferative in vitro, and to be the only antiestrogenic compound so far known to induce long-term regression of MCF-7 tumours implanted into nude mice. In order to obtain more insight into the therapeutic potential of this molecule, we performed a new set of experiments in vitro and in vivo in comparison with tamoxifen and/or ICI 182,780. In vitro, 1 nM RU 58668 induced an accumulation of MCF-7 cells in G0/G1 phases of the cell cycle within 48 h and, in contrast to trans-4-hydroxy-tamoxifen, blocked the invasiveness of ras-transfected MCF-7 cells into the chick embryo heart during the three weeks of co-culture. An in vivo dose-effect relationship study showed that RU 58668 induced a regression of MCF-7 tumour with as low a dose as 10 mg/kg/week, and that such an effect can not be obtained either with a sublethal dose of adriamycin or with ICI 182,780, (2-250 mg/kg/week). This reduction in the tumour volumes accords with histological modifications of the tumours, which showed a decrease in the ratio of epithelial cells over the tumoral mass, and with a concomitant decrease in their regrowth potential when reimplanted into naive nude mice. Taken together, these results suggest a promising usefulness for RU 58668 in the treatment of metastatic breast cancer in women.

Exploration of the therapeutic potential of the antiestrogen RU 58668 in breast cancer treatment.

The recently described pure antiestrogen RU 58668 displayed potent antiproliferative activities in vitro on several ER+ human mammary cell lines, stimulated either by estradiol or by endogenous or exogenous growth factors. Moreover, when administered to nude mice it proved to be the only antiestrogen to induce regression (at least 10 weeks) of estradiol-stimulated MCF-7 tumors, whereas tamoxifen only stabilized the tumor volume for 4 to 8 weeks. So the first purpose of this work was to study the effect of RU 58668 for 6 months and to evaluate its activity on tumors which escaped from the tamoxifen treatment. On the other hand, we looked for its effect on models more related to frequently described clinical observations, such as the overexpression of an oncogene or the implication of autocrine or paracrine growth factors. Long-term studies of RU 58668 on the estradiol-stimulated MCF-7 model showed that this compound induced a shrinking of tumor volumes for at least 25 weeks (3 to 6 times longer than the stabilization induced by tamoxifen) and was able to reduce the volume of tumors which escaped from, or even were stimulated by, tamoxifen. On models of spontaneously growing tumors, where the overexpression of an oncogene or the production of growth factors was involved, RU 58668 induced the same tumor shrinking that was previously observed on estradiol- or tamoxifen-stimulated models. Finally, when MCF-7 cells were injected in the uteri, a spontaneous tumor take was observed (in about 80-90% of the animals), leading to a more than twofold increase in uterus weight. This growth is largely stimulated by estradiol and tamoxifen. On this model, histological examination showed that only 30% of the animals receiving RU 58668 displayed tumoral microfoci. These studies suggest that RU 58668 may be used for the treatment of ER+ patients which are primarily resistant to or which escaped from tamoxifen treatment. Its preventive activity on tumor take also suggests its use as an adjuvant to prevent the development of metastases.

11 beta-Amidoalkoxyphenyl estradiols, a new series of pure antiestrogens.

11 beta-Amidoalkoxyphenyl estradiols, a series of new antiestrogens, have been prepared and compared with tamoxifen (TAM) and 4-hydroxytamoxifen (OH-TAM). In vitro, these compounds were up to 20 times as active as OH-TAM on estradiol (E2)-stimulated MCF-7 cells. Unlike TAM or OH-TAM which were inactive, they displayed potent growth inhibitory effects on MCF-7 cells stimulated by a cocktail of epidermal growth factor and platelet derived growth factor. One of the most active compounds, 5e, was tested in vivo for its antiuterotrophic and antitumoral activities: it proved to be fully antiuterotrophic at 3 mg/kg subcutaneously in mice while being devoid of any uterotrophic activity. It inhibited the E2-induced growth of MCF-7 tumors implanted in nude mice and prevented the partial agonistic activity of TAM on MCF-7 tumor growth in ovariectomized mice. Moreover, on MCF-7 variant tumors, 5e, unlike TAM, did not display any proliferative activity, but inhibited the TAM-induced growth. Overall, these results show that this new series of compounds displays an improved activity profile compared with that of TAM, on tests relevant to human breast cancer treatment.

RU 58,668, a new pure antiestrogen inducing a regression of human mammary carcinoma implanted in nude mice.

RU 58,668, a new steroidal antiestrogen, has been synthesized. Its in vitro and in vivo pharmacological activities have been compared to those of tamoxifen and ICI 182,780. In vitro, it displayed affinities for human and murine estrogen receptors equivalent to those of 4-hydroxy-tamoxifen, along with moderate affinities for progestin and glucocorticoid receptors. RU 58,668 proved to be a potent antiproliferative agent on MCF-7 cells stimulated by estradiol, or by exogenous or endogenous growth factors (IC50, 0.01 nM). It also inhibited the growth of the insulin-stimulated T47D cell line. In vivo, RU 58,668 displayed a total anti-uterotrophic activity in mice or rats without exhibiting any agonistic effect. Moreover, RU 58,668 was the only antiestrogenic compound tested so far to be able to induce a long term regression of human mammary MCF-7 tumors implanted in nude mice, suggesting its potential use for the treatment of advanced breast cancer.

11 beta-amidoalkyl estradiols, a new series of pure antiestrogens.

In order to find new antiestrogens, devoid of any agonistic activity, a series of 11 beta-amidoalkyl estradiols were prepared. These compounds have been studied in comparison with tamoxifen (TAM): in vitro, for their relative binding affinities (RBA) for mouse and MCF-7 estrogen receptors (ER) and for their antiproliferative effect on MCF-7 (estradiol or EGF/PDGF stimulated) and Ly2 human breast cancer cell lines; in vivo, for their uterotrophic/antiuterotrophic activities in the mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice. The most representative compounds are N-methyl-N-isopropyl-(3,17 beta-dihydroxy-estra-1,3,5(10)-trien-11 beta-yl)- undecanamide (RU 51625) and its 17 alpha-ethynyl derivative (RU 53637). They showed good RBAs for ER and a stronger antiproliferative effect than TAM in vitro. Unlike TAM, these compounds inhibited growth factor stimulated MCF-7 proliferation, and the growth of the TAM resistant cell line Ly2. In vivo, they were completely devoid of uterotrophic activity, when given subcutaneously in mice, but exhibited a slight agonistic effect when administered orally. They showed interesting antitumor activities in nude mice by the percutaneous route, but RU 53637 was significantly more potent than RU 51625 when given orally.

New analogues of mifepristone with more dissociated antiprogesterone activities.

Mifepristone (RU 486 or RU 38486) possesses strong antiprogesterone and antiglucocorticoid along with moderate antiandrogen properties, which would limit its use in some therapeutic applications. In a search for more dissociated derivatives, the hydroxy substituent and the propynyl group in position 17 of the RU 486 series was replaced by a spiroether group, which is known to induce specific affinity for the progestin receptor in steroid series. The substituents in the para position of the 11 beta-phenyl group, leading to the most potent derivatives in the RU 486 series, were retained. The new derivatives have been studied in vitro for their relative binding affinities (RBAs) for the steroid receptor and in vivo for their hormonal and antihormonal activities. The selected compounds, RU 46556 and RU 49295 display the following properties: in vitro, like RU 486, they show a strong RBA for the rabbit progestin receptor, but a much lower one for the rat thymus glucocorticoid receptor; in vivo they are about three times more active than RU 486 for inducing abortion in rats, but unlike the latter they are devoid of any antiglucocorticoid activity on the thymus weight in rats. These antiprogesterone effects have been confirmed on the deciduoma formation in rats and on the endometrial proliferation in rabbits. However, in contrast to RU 486 in the latter test, some progestomimetic activity has been observed. RU 46556 and RU 49295 are now under extensive pharmacological study.

Synthesis of new fluorescent spirolactone derivatives: determination of their affinities for aldosterone receptors.

Fluorescent spirolactone derivatives are obtained by coupling 3-carboxylic coumarins to a spirolactone bearing a 3-hydroxypropyl chain in the 7 alpha position. The two esters prepared by this method are highly fluorescent (emission 383 and 408 nm).